Multiyear Factor VIII Expression after AAV Gene Transfer for Hemophilia A.

BACKGROUND: The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose. METHODS: In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5 × 1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII. RESULTS: The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration). CONCLUSIONS: Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.).

Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer.

Gene therapy with adeno-associated virus (AAV) vectors has demonstrated safety and long-term efficacy in a number of trials across target organs, including eye, liver, skeletal muscle, and the central nervous system. Since the initial evidence that AAV vectors can elicit capsid T cell responses in humans, which can affect the duration of transgene expression, much progress has been made in understanding and modulating AAV vector immunogenicity. It is now well established that exposure to wild-type AAV results in priming of the immune system against the virus, with development of both humoral and T cell immunity. Aside from the neutralizing effect of antibodies, the impact of pre-existing immunity to AAV on gene transfer is still poorly understood. Herein, we review data emerging from clinical trials across a broad range of gene therapy applications. Common features of immune responses to AAV can be found, suggesting, for example, that vector immunogenicity is dose-dependent, and that innate immunity plays an important role in the outcome of gene transfer. A range of host-specific factors are also likely to be important, and a comprehensive understanding of the mechanisms driving AAV vector immunogenicity in humans will be key to unlocking the full potential of in vivo gene therapy.

The Effect of CpG Sequences on Capsid-Specific CD8+ T Cell Responses to AAV Vector Gene Transfer.

Transfer of genes by adeno-associated virus (AAV) vectors is benefiting patients with particular genetic defects. Challenges remain by rejection of AAV-transduced cells, which may be caused by CD8+ T lymphocytes directed to AAV capsid antigens. Reducing the number of CpG motifs from the genome of AAV vectors reduces expansion of naive T cells directed against an epitope within the capsid. In contrast, AAV capsid-specific memory CD8+ T cells respond more vigorously to AAV vectors lacking CpG motifs than to those with CpG motifs presumably reflecting dampening of T cell expansion by cytokines from the innate immune system. Depending on the purification method, AAV vector preparations can contain substantial amounts of empty AAV particles that failed to package the genome. Others have used empty particles as decoys to AAV-neutralizing antibodies. We tested if empty AAV vectors given alone or mixed with genome-containing AAV vectors induce proliferation of naive or memory CD8+ T cells directed to an antigen within an AAV capsid. Naive CD8+ T cells failed to respond to empty AAV vectors, which in contrast induced expansion of AAV-specific memory CD8+ T cells.

Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 × 1010 to 2 × 1012 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

Changes in chromatin state reveal ARNT2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness.

Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients' glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients' tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.

Complement System Response to Adeno-Associated Virus Vector Gene Therapy.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

AF1q/MLLT11 regulates the emergence of human prothymocytes through cooperative interaction with the Notch signaling pathway.

The mechanisms regulating the emergence of BM prothymocytes remain poorly characterized. Genome-wide transcriptome analyses looking for genes expressed in human prothymocytes led to the identification of AF1q/MLLT11 as a candidate gene conceivably involved in this process. Analysis of AF1q protein subcellular localization and intracellular trafficking showed that despite pronounced karyophily, it was subjected to constitutive nuclear export followed by ubiquitin-mediated degradation in the centrosomal area. Using in vitro assays based on either forced expression or shRNA-mediated silencing of AF1q, we provide evidence that the protein promotes T- over B-cell differentiation in multipotent hematopoietic progenitors. At the molecular level, AF1q confers to multipotent progenitors an increased susceptibility to Delta-like/Notch-mediated signaling. Consistent with these findings, enforced AF1q expression in humanized mice fosters the emergence of BM CD34(+)CD7(+) prothymocytes, enhances subsequent thymus colonization, and accelerates intrathymic T-cell development. In contrast, AF1q silencing provokes a global shift of BM lymphopoiesis toward the B-cell lineage, hinders prothymocyte development, inhibits thymus colonization, and leads to intrathymic accumulation of B cells. Our results indicate that AF1q cooperates with the Notch signaling pathway to foster the emergence of BM prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage.

The Effect of Rapamycin and Ibrutinib on Antibody Responses to Adeno-Associated Virus Vector-Mediated Gene Transfer.

Adeno-associated virus (AAV) vector-mediated gene transfer is lessening the impact of monogenetic disorders. Human AAV gene therapy recipients commonly mount immune responses to AAV or the encoded therapeutic protein, which requires transient immunosuppression. Most efforts to date have focused on blunting AAV capsid-specific T cell responses, which have been implicated in elimination of AAV-transduced cells. Here, we explore the use of immunosuppressants, rapamycin given alone or in combination with ibrutinib to inhibit AAV vector- or transgene product-specific antibody responses. Our results show that rapamycin or ibrutinib given alone reduces primary antibody responses against AAV capsid, but the combination of rapamycin and ibrutinib is more effective, blunts recall responses, and reduces numbers of circulating antibody-secreting plasma cells. The drugs fail to lower B cell memory formation or to reduce the inhibitory effects of pre-existing AAV capsid-specific antibodies on transduction efficiency.

Pre-existing humoral immunity and complement pathway contribute to immunogenicity of adeno-associated virus (AAV) vector in human blood.

AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor-like state.

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL-60, U266) derived from hematopoietic malignancies contain not only CD34(-) but also CD34(+) subpopulations. The CD34(+) cells displayed a stem/progenitor-like phenotype since, in contrast to CD34(-) cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose-based medium. Strikingly, a constant fraction of the CD34(+) and CD34(-) cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz-scid/scid mice. Cultures in vitro showed that the proportion of CD34(+) stem/progenitor-like cells in the population was decreased by cell-cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34(+) cells and overall colony-forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34(+) cells can provide an in vitro model for studies of cancer stem/progenitor cells.

In acute myeloid leukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is induced by TLR ligands and interferon-gamma and can be reversed using MEK inhibitors.

B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model of leukemia. B7-H1 can be constitutively expressed by cancer cells, but is also induced by various stimuli. Therefore, we examined the constitutive and inducible expression of B7-H1 and the consequences of this expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. In addition, we studied blast cells after incubation with interferon-gamma or toll-like receptors (TLR) ligands. Finally, we evaluated functionality of cytotoxic T-cell activity against blast cells. Expression of B7-H1 upon diagnosis was high in 18% of patients. Expression of TLR2, 4 and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN-γ induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced upon relapse in some patients. MEK inhibitors, including UO126 and AZD6244, reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN-γ or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs.

The isoprenoid pathway and transcriptional response to its inhibitors in the yeast Saccharomyces cerevisiae.

This review presents new insights into the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae, in particular the short-term transcriptional response to two inhibitors, lovastatin and zaragozic acid (ZA). Whereas lovastatin blocks whole isoprenoid pathway, ZA only blocks the sterol branch. Consequently, their effects on the cellular level of farnesyl diphosphate (FPP) are different. Lovastatin decreases the FPP level, whereas ZA, by inhibiting the main FPP-consuming enzyme, increases FPP availability in the cell. We discuss the role of genes whose expression is affected by both inhibitors and consider possible association of these genes with the regulation of the isoprenoid pathway.

The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae.

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis-prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7Delta cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.

In Vitro Expansion of Anti-viral T Cells from Cord Blood by Accelerated Co-cultured Dendritic Cells.

Hematopoietic stem cell transplantation (HSCT) using unrelated cord blood (CB) donors is a suitable approach when an HLA-matched donor is not available. However, one important drawback is the risk of life-threatening viral infections prior to immune reconstitution, particularly from adenoviruses (AdVs). Although adoptive therapy with ex vivo expanded virus-reactive donor T cells has proven effective to treat these infections in HSCT recipients, the manufacturing process is complex and requires large numbers of cells, which is incompatible with CB donor units. Here, we have adapted our previous accelerated co-cultured dendritic cell (acDC) method, which allows to efficiently and rapidly expand peripheral blood T cells reactive to a given antigen, for use on limited CB material. Selected cytokine cocktails induced DC differentiation and maturation from unfractionated CB mononuclear cell cultures and simultaneously stimulated and expanded, within 10 days, functional CD8+ T cells specific for the model antigen MelanA or AdV immunodominant peptides. In addition, the use of G-Rex cultures yielded numbers of AdV-reactive CD8+ T cells compatible with adoptive cell therapy applications. Our acDC strategy, which uses reagents compatible with good manufacturing practices, may be promptly translated into the clinic for treating intercurrent infections in CB HSCT recipients.

Exposure to wild-type AAV drives distinct capsid immunity profiles in humans.

Recombinant adeno-associated virus (AAV) vectors have been broadly adopted as a gene delivery tool in clinical trials, owing to their high efficiency of transduction of several host tissues and their low immunogenicity. However, a considerable proportion of the population is naturally exposed to the WT virus from which AAV vectors are derived, which leads to the acquisition of immunological memory that can directly determine the outcome of gene transfer. Here, we show that prior exposure to AAV drives distinct capsid immunity profiles in healthy subjects. In peripheral blood mononuclear cells (PBMCs) isolated from AAV-seropositive donors, recombinant AAV triggered TNF-α secretion in memory CD8+ T cells, B cell differentiation into antibody-secreting cells, and anti-capsid antibody production. Conversely, PBMCs isolated from AAV-seronegative individuals appeared to carry a population of NK cells reactive to AAV. Further, we demonstrated that the AAV capsid activates IL-1ß and IL-6 cytokine secretion in monocyte-related dendritic cells (moDCs). IL-1ß and IL-6 blockade inhibited the anti-capsid humoral response in vitro and in vivo. These results provide insights into immune responses to AAV in humans, define a possible role for moDCs and NK cells in capsid immunity, and open new avenues for the modulation of vector immunogenicity.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.

The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8186-194 (ZnT8186-194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186-194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186-194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186-194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186-194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation.

The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.

Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients.

Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.